Pe siglec f

Chest wall exposure was conducted between the third and seventh ribs, and a cover glass slide was adhered to the lung allograft using tissue glue (VetBond) applied in a gentle manner as not to disturb blood flow. AMs were imaged with PE-Siglec F (2 μg, clone S17007L; BioLegend) administered i.v. 30 minutes after engraftment. FACS-sorted PD-1⁺CD49a⁺CD69⁺CD8⁺ T cells isolated from e3T-FVB allografts were labeled with 5 μM CFSE, and between 1 and 3 × 10⁵ cells were intratracheally administered 1 day before imaging. Data were collected by sequential z sections (24, 2.5 μm each), which were acquired in an imaging volume of 200 × 225 × 60 μm³. Analyses were performed with Imaris (Bitplane). Associations between AMs and TRM cells were defined as physical interactions that lasted longer than 15 seconds. For each lung transplant, at least 5 areas were examined up to approximately 50 μm deep. Data shown has been pooled from at least 3 mice per group.

BALF was centrifuged at 300g, 5 min. The supernatant was stored at − 80 °C until analysis. Cell sediments were resuspended with PBS and stained with APC/Cyanine7 anti-CD45, PE/Cyanine7 Ly6G, APC-CD11c, and PE-Siglec-F, which were purchased from BioLegend (San Diego, CA, USA) or eBioscience (San Diego, CA, USA). Eosinophils were identified as CD45⁺ Ly6G⁻ CD11c⁻ Siglec-F⁺, neutrophils as CD45⁺ Ly6G⁺, and alveolar macrophages as CD45⁺ Ly6G⁻ CD11c⁺Siglec-F⁺. Hemocytometer was used to count total cells in BAL.